Purified and modified hemoglobins are potential blood substitutes, however significant toxicities are associated with their in vivo application. We have studied the effects of purified hemoglobin A0 (HbA0) (10nm -10uM) on platelet reactivity. HbA0 did not activate platelets when added alone but potentiated agonist-induced platelet aggregation in a concentration dependent manner. HbA0 (10uM) nearly doubled the extent of platelet aggregation induced with submaximal concentrations of collagen, thrombin and arachidonic acid but did not potentiate aggregation induced with ADP or ADP/epinephrine. The potentiation was not blocked by indomethacin, apyrase, yohimbine, superoxide dismutase or catalase. Heme-mediated reactions only partially accounted for this potentiation since methemoglobin A0, carbon monoxide-treated HbA0 and cyanomethemoglobin A0 potentiated aggregation 62-97% as well as native hemoglobin A0. Affinity chromatography of solubilized platelet membranes on hemoglobin-agarose revealed an interaction of hemoglobin with a 125 kDa and a 95 kDa protein. These proteins were identified as the aIIb3 integrin complex (glycoprotein IIb/IIIa) by immunoblotting with monoclonal antibodies. This interaction was prevented with free HbA0 in solution suggesting a specific interaction. Hemoglobin can convert arachidonic acid into prostaglandins E2 and F2a in an indomethacin-independent manner. These prostaglandins can interact with platelets through the thromboxane receptor. Blocking of this receptor with antagonists or removal of these prostaglandins as they are produced with monoclonal antibodies, prevents the hemoglobin potentiation of aggregation. Thus, the potentiation of aggregation is mediated by prostaglandins produced by hemoglobin. The significance of this project lies in identifying interactions between circulating blood cells and hemoglobin-based blood substitutes in vitro and thus predict potential toxic reactions when the blood substitutes are used in vivo.